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Simultaneous separation and purification of mononuclear and polymorphonuclear cells from the peripheral blood of cats
1Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg 24061.
Insights
A new method using double-density centrifugation effectively separates feline peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNC) from the same blood sample, aiding immunodeficiency disease research.
Area of Science:
- Veterinary Immunology
- Cell Biology
- Feline Infectious Diseases
Background:
- Peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNC) are crucial in feline immunodeficiency diseases like those caused by FeLV and FIV.
- Simultaneous isolation of both PBMC and PMNC from a single feline blood sample is advantageous for comparative and functional studies.
Purpose of the Study:
- To evaluate and optimize methods for the simultaneous separation and purification of PBMC and PMNC from feline blood.
- To identify the most effective technique for obtaining distinct, high-purity populations of both cell types from the same sample.
Main Methods:
- Comparative analysis of cell separation techniques including lysed whole blood, single-density Histopaque centrifugation (1.077 or 1.119), and double-density centrifugation (1.077/1.119).
- Flow cytometric analysis of cell populations and CD4/CD8 T-cell ratios in purified lymphocytes.
- Evaluation of consistency, practicality, and effectiveness of each method.
Main Results:
- Histopaque 1.077/1.119 double-density centrifugation of diluted whole blood proved to be the most consistent, practical, and effective method.
- This method yielded highly purified PBMC (average 86%) and PMNC (average 98%) as separate entities.
- Isolated lymphocytes exhibited an average CD4/CD8 T-cell ratio of 2.0.
Conclusions:
- The Histopaque 1.077/1.119 double-density gradient centrifugation method enables efficient purification and physical separation of lymphocytes and phagocytes from feline blood.
- This technique allows simultaneous examination of both PBMC and PMNC from a single sample, facilitating research into feline immunodeficiency.
- This method is valuable for comparative immunological studies in cats.
Abstract:
Peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNC) play important roles in immunodeficiency diseases and AIDS-like syndromes in cats caused by feline leukemia virus (FeLV) and presumably also when caused by feline immunodeficiency virus (FIV). For comparative or functional studies it is advantageous or necessary to obtain these cells as separate entities from the same sample of an animal. Therefore, we analyzed the technical parameters of obtaining, separating and purifying these cells simultaneously from several blood samples of several cats. Flow cytometric studies with outbred cats indicated that of various cell separation/purification methods, e.g. from lysed whole blood, by Histopaque 1.077 or 1.119 single-density, or by 1.077/1.119 double-density centrifugation, the 1.077/1.119 double-density centrifugation of diluted whole blood is the most consistent, practical and effective method of yielding both highly purified PBMC and PMNC as separate entities from the same sample. The interface between plasma and 1.077 contained an average 86% PBMC vs. 14% PMNC, and the interface between 1.077 and 1.119 an average of 2% PBMC vs. 98% PMNC. Lymphocytes separated by this method had an average CD4/CD8 T-cell ratio of 2.0. These data indicate that Histopaque 1.077/1.119 double-density gradient allows purification and physical separation of lymphocytes and phagocytes from a blood sample, enabling the investigator to examine both cell types from the same sample simultaneously.